The present invention relates to a method for determining the presence of an analyte by means of an energy transfer that results in the generation of bathochromic and/or delayed fluorescence emission. Fluorescence radiation, emitted from a first energy emitter (E.sub.1), is absorbed by a second energy emitter (E.sub.2). This second energy emitter emits fluorescence radiation of a longer wavelength than the first energy emitter. The second energy emitter may in addition emit fluorescence for a substantially longer period than the first energy emitter (in a delayed manner). The detection of either the bathochromic fluorescence or of any fluorescence after a time period during which fluorescence radiation from background sources has decayed verifies the presence of the analyte.
Methods for the in-vitro detection of analytes are well known in the art. The methods include the formation of antibody-antigen complexes (immunodetection), and the formation of nucleic acid complexes (polynucleotide hybridization). The analyte can be an intact cell or a component of the cell. Examples of analytes are bacteria, viruses, antigens, antibodies, and polynucleotides.
The immunoassay for detecting antigen (or antibody) analytes is well established in the art. The assay involves the formation of antigen-antibody complexes. In radioimmunoassay (RIA), a radioactive isotope is used to report the presence of the analyte. In enzyme immunoassay, chromogen or fluorescence generated by means of an enzyme is used to report the presence of the analyte. Several enzyme immunoassays are currently in use. They include the enzyme multiplied immunoassay technique (EMIT) and the enzyme-linked immunosorbent assay (ELISA). The ELISA method comprises the "sandwich" technique for antigen, the antibody assay, and the competitive assay for antigen.
A typical ELISA assay using the sandwich technique is carried out by adsorbing an antibody to the surface of a support. The test specimen is added to the support and the antigen allowed to complex to the antibody. Unbound antigen is washed away. An enzyme-conjugated antibody is added and allowed to react with a different set of determinants on the bound antigen which are not blocked by the support-absorbed antibody. After the reaction, the excess of unbound enzyme-linked antibody is washed away and a substrate of the enzyme is added to the support. The generation of a colored product indicates the presence of the antigen in the test specimen. See Enzyme Immunoassays by S. Bakerman in Laboratory Management, August 1980, p. 21.
A drawback of these methods is that they cannot be carried out in one-step, to achieve detection, i.e., by adding the antibody to the antigen or the antigen to the antibody. One or more washing steps are required to remove antibody unbound to antigen (or vice versa). Also, a number of these methods involves competition kinetics which in some instances can provide ambiguous results.
Polynucleotide hybridization assays using a polynucleotide probe for verifying the presence of a target polynucleotide analyte is a well known method. Hybridization is based on complementary base-pairing.
When single-stranded polynucleotide probes are incubated in solution with single-stranded target polynucleotides that are immobilized on a support, complementary base sequences pair to form double-stranded hybrid molecules. The double-stranded hybrid molecules remain immobilized on the support while unbound polynucleotide probe molecules are washed off. See M. Grunstein and J. Wallis, METHODS IN ENZYMOLOGY, volume 68, R.W.U (Ed) (1979) pp. 379-469; A. R. Dunn, and J. Sambrook, METHODS IN ENZYMOLOGY, volume 65; part 1, (1980) pp. 468-478; Modified Nucleotides And Methods Of Preparing And Using The Same by D. C. Ward, A. A. Waldrop, and P. R. Langer, European Patent Publication No. 0,063,879 published Nov. 3, 1982; DNA Probes for Infectious Disease by A. J. Berry and J. B. Peter, Diagnostic Medicine (March, 1984) pp. 1-8; and Recombinant DNA Technology: Some Applications In Clinical Microbiology by Wie-Shing Lee and James L. Bennington, Laboratory Management (April, 1985) pp. 21-26.
The polynucleotide probes generally comprise a polynucleotide segment and a signalling segment which is attached to the polynucleotide. The polynucleotide segment of the probe has the ability to base-pair, i.e. hybridize to a sequence of interest, namely the analyte or target polynucleotide. The signalling segment of the probe has or produces the means by which the presence of the analyte moiety can be verified. The means can be, for example, fluorescence, phosphorescence, radioactivity, chromogen, or electron density.
The method of detecting the presence of a target polynucleotide generally involves several steps, one of which is the separation of hybridized polynucleotide probe from unhybridized probe. The separation can be facilitated by immobilizing either the probe or the target onto a solid support. Typically, double-stranded polynucleotides are isolated from a sample suspected of containing a target polynucleotide. The double-stranded polynucleotides are cut into smaller segments by means of restriction endonuclease enzyme digestion, the segments are separated by gel electrophoresis, and the segments are transferred from the gel onto a support, for example, nitrocellulose paper. Alternatively, the double-stranded polynucleotides are fixed directly onto the support without any prior enzyme digestion. The fixed polynucleotides are contacted with a solution containing the polynucleotide probe, and the support is heated to about 80.degree.-90.degree. C. to denature the polynucleotide double-strands. (The double-strands can alternatively be denatured by means of alkali). The system, which now contains the denatured target polynucleotide and the polynucleotide probe, is allowed to cool to an appropriate temperature to allow hybridization to take place. After sufficient time has elapsed for hybridization to be complete, which can be for ten minutes to several hours, the fixed target polynucleotide is washed to remove all unbound polynucleotide probes. The signalling moiety of the polynucleotide probe is now detected, either directly, for example, by means of radioactivity or fluorescence, or indirectly, for example, by means of a chromogen formed through an enzymatic reaction.
A drawback of this method is that it requires several steps before the presence of the target polynucleotide can be verified. Namely, it requires the fixation of the target polynucleotide to a support, the contacting of the target polynucleotide with a polynucleotide probe, and the removal of all unhybridized polynucleotide probes from the support. Besides being time consuming, the method is not readily amenable to automation and requires some expertise for obtaining reproducible results. In addition, hybridization and detection of the target polynucleotide in a one phase system is not possible.
One method seeking to overcome the above drawbacks by detecting the presence of a target polynucleotide with a homogenous (one-step or one phase) nucleic acid hybridization assay has been reported. The method comprises hybridizing first and second single-stranded polynucleotides, both of which contain light-sensitive labels, with a complementary single-stranded polynucleotide target from a sample such that non-radiative energy transfer occurs between the light-sensitive labels of the first and second polynucleotides. At least one of the light-sensitive labels is of the absorber/emitter type such that energy absorbed by this label from the emission of the other light-sensitive label is reemitted at a different wavelength. These secondary emissions can only occur if hybridization of both the first and second single-stranded polynucleotides to the target polynucleotide has taken place. The quantity of the target polynucleotides in the sample is related to the amount of secondary light emitted. See European Patent Publication No. 0,070,685 by Michael James Heller, published Jan. 26, 1983.
A drawback of this method is that it requires two separate polynucleotide strands to detect the presence of a target polynucleotide. In addition, the method requires the presence of a chemiluminescent catalyst, an absorber/emitter moiety, and chemiluminescent reagents effective for causing light emission in the presence of the chemiluminescent catalyst. Furthermore, only one label can be attached per polynucleotide probe because the light-sensitive label is attached to the sugar moiety of a terminal nucleoside. Also, the bulky labels may prevent hybridization of the bases adjacent to the labels.
Another method for detecting the presence of a target polynucleotide by means of a homogeneous assay has been recently reported. The method involves forming a hybrid between the target polynucleotide and the polynucleotide probe, wherein the hybrid has binding sites for two specific binding reagents, one of which comprises a first label and the other a second label. The interaction of the first and second labels provide a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid, as compared to when the two labeled reagents are not so bound. The formation of the hybrid assay product brings the two labels within approximate interaction distance of one another, e.g., as in the cases of sequential catalyst (enzyme) interaction and energy transfer. Since the labels provide a response which is distinguishable when the labels are associated with a hybridized probe, no separation step is required. See European Patent Application No. 0,144,914 by James P. Albarella et al., published Nov. 29, 1984.
The method has two main embodiments. The first embodiment involves the generation of a component which subsequently produces a color. This embodiment has a drawback in that it requires the use of two distinct chemical reactions, namely, the reaction of the first label to produce a diffusible mediator product, and the reaction of the mediator product with the second label to yield a detectable product. In addition, detection depends on the formation and maintenance of a higher localized concentration of the mediator product in the vicinity of the first label as compared to elsewhere in the solution. Furthermore, both reactions require the use of bulky enzyme molecules attached to the polynucleotide probe. These bulky molecules may sterically "clash" with each other.
A second embodiment involves that of energy transfer, namely the emission of photons from a first label, for example, fluorescence, followed by absorption of the photons by a second label, to either quench the emission, or to provide a second emission. This has a drawback in that when an intercalator is the first label, it is attached to the polynucleotide probe covalently. In addition, the method requires the formation of two complexes, namely the formation of a polynucleotide/polynucleotide complex, and the formation of an antigen/antibody complex. Furthermore, one aspect involves the quenching of emitted photons, and since hybridization of probe to target is usually no more than a few percent, such minute quenching would produce ambiguous results.
Fluorescence detection is widely used in hybridization assays. In fluorescence spectroscopy the substance to be determined which is present in a liquid or a solid phase is subjected to a radiation with a known spectral distribution, for instance light with a limited band width. The fluorescent radiation thereby emitted has a longer wavelength than the exciting radiation and this radiation is specific for the substance to be determined. The measurement of the intensity of the fluorescent radiation constitutes a quantification of the substance to be determined. Fluorescent moieties attached to polynucleotide probes are most efficient when they have a high intensity, a relatively long emission wavelength (more than 500 nm), a high Stoke's shift, and the ability to be bound covalently to a polynucleotide probe without negatively affecting its hybridization capabilities. Aromatic agents used in biological systems that give a rather strong fluorescence and are relatively stable include, for example, fluorescenisothiocyanate (FITC), rhodamines (RBITC, TRITC, RB-200-SC), dansil chloride (DNS-Cl), and fluorescamine (FL).
Fluorescence is generally measured with a spectrofluorimeter. A disadvantage of current methods for detecting signalling moieties with spectrofluorimeters is that the detection sensitivity is limited because of interfering fluorescence or noise in the exciting and detecting systems that increases the background. Interfering fluorescence is generated from substances such as substrate molecules, non-specifically bound compounds, sample holders, air particles, and the intrinsic fluorescence of the biological systems. The background is also affected by a heavy scattering which gives rise to an interference, especially when aromatic organic agents with a small Stoke's shift (less than 50 nm) are used.
Several approaches have been described that attempt to overcome the background problem with fluorescence detection. One approach, described in U.S. Pat. No. 4,058,732, measures delayed fluorescence using a signalling moiety comprising a substance with a fluorescence emission having a duration that considerably exceeds the duration of the fluorescence of the noise sources. A laser pulse is used to excite a sample, and the detection of the fluorescence from the signalling moiety takes place only when a sufficiently long time has passed for the fluorescence from the noise sources to have decayed. This method has drawbacks in that it is not readily adaptable to commercial use, and is not amenable for a homogenous assay.
A second approach, described in U.S. Pat. No. 4,374,120, by E. Soini and I. Hemmilia, discloses a method for determining the presence of an antigen by attaching a first ligand to an antibody, complexing a lanthanide metal to the first ligand, and complexing a second ligand to the lanthanide metal. The antigen-containing sample is fixed to a support, antibodies are then contacted with the sample, and unbound antibodies are washed away. A radiation pulse of short duration is used to excite the second ligand. Energy is transfered from the triplet state of this ligand to the chelated metal which emits radiation at a longer wavelength and for a longer time period than the noise sources. Detection of this delayed fluorescence verifies the presence of the antigen. This method has a drawback in that it cannot be carried out in one step; all unbound antibodies must be washed away from the support.